500μg of each peptide, in lysis buffer, was bound to streptavidin-conjugated magnetic sepharose beads (Streptavidin-MagSepharose, GE Healthcare) by incubation at 4°C on a rotating wheel for 1h, then washed three times with lysis buffer.
The cell extract was pre-cleared by incubation with empty streptavidin conjugated magnetic beads at 4°C on a rotating wheel for 1h.
After removal of the pre-clearing beads, the extract was incubated at 4°C on a rotating wheel for a further 2h with each of the biotinylated peptides bound to streptavidin-conjugated magnetic sepharose beads.
The beads were washed three times with lysis buffer without protease inhibitors, transferred to fresh eppendorf tubes and washed twice more with lysis buffer without either protease inhibitors or Triton-x-100.
5% of the beads were taken for western blot analysis and the remainder were subjected to trypsin-digest and the products analysed by mass spectroscopy, as described previously [45 (link)].
The cell extract was pre-cleared by incubation with empty streptavidin conjugated magnetic beads at 4°C on a rotating wheel for 1h.
After removal of the pre-clearing beads, the extract was incubated at 4°C on a rotating wheel for a further 2h with each of the biotinylated peptides bound to streptavidin-conjugated magnetic sepharose beads.
The beads were washed three times with lysis buffer without protease inhibitors, transferred to fresh eppendorf tubes and washed twice more with lysis buffer without either protease inhibitors or Triton-x-100.
5% of the beads were taken for western blot analysis and the remainder were subjected to trypsin-digest and the products analysed by mass spectroscopy, as described previously [45 (link)].
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